Prolonged survival for patients diagnosed with glioblastoma multiforme (GBM) is exceedingly rare with approximately 2% of patients surviving beyond 5 years. Approximately a third of patients with prolonged survival have a more indolent sub-type of GBM governed by mutation of isocitrate dehydrogenase, but the remainder of long survivors has tumors that are typically associated with less than a 6 month median initial progression-free survival (PFS) following aggressive surgery, radiation (RT) and temozolomide (TMZ) therapy. The focus of this application is to define the molecular mechanisms that result in extended initial PFS in these rare patients with non-IDH mutant tumors. To study the spectrum of treatment efficacy in GBM, the Mayo Brain Tumor SPORE has developed a panel of 43 primary tumor xenografts derived from patients with newly diagnosed primary GBM. Three of these models (GBM5, 75 and 84) were derived from patients with long initial PFS, and each demonstrates unique hypersensitivity to cytotoxic therapy. These results support the hypothesis that prolonged PFS, in at least some patients, results from a unique sensitivity to initial therapy and not just from a generally indolent tumor biology. In this application, patient tumors and xenograft models will be analyzed with next generation sequencing (NGS) and functional proteomic tools, and then the xenograft models will be manipulated to robustly define mechanisms of hypersensitivity to RT and TMZ therapies. In Aim 1 a collection of 15 long-surviving patient samples will be analyzed by whole exome seq (WES) and mRNAseq, and paired patient/xenograft samples will be subjected to a more detailed molecular landscape analysis with whole genome seq and epigenomic profiling. Through an integrated analysis of these data and comparison to WES/mRNAseq data from short survivors in TCGA, mechanistic hypotheses linked to prolonged disease control will be generated that will be tested in subsequent aims. Aim 2 specifically will test mechanisms of extreme therapy sensitivity in the GBM5, 75 and 84 xenograft models. Integrated NGS and functional proteomic analyses will define putative mechanisms of hypersensitivity, and manipulation of gene expression then will be used to define the importance of specific pathways on therapy response. The ultimate goal of this application is to translate an understanding of molecular mechanisms associated with prolonged disease control into novel therapeutic strategies that could significantly enhance the efficacy of therapy for typical GBM tumors. Thus, in Aim 3 we will use the insights gained from Aims 1 and 2 to design a custom shRNA library, and then use an in vivo shRNA screen and subsequent validation studies to define pathway targets that can increase significantly the efficacy of radiation and/or temozolomide.